Chicken liver 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase has been purified to homogeneity. The catalytic and kinetic properties of the purified enzyme has been investigated and compared with rat liver HMG-CoA reductase. Both rat and chicken liver HMG-CoA reductase activity was inhibited by ATP plus MG to the plus 2 power. The inactivation of HMG-CoA reductase activity was time, temperature, and ATP concentration dependent. Incubation of microsomal HMG-CoA reductase with (gamma to the negative 32 power P) ATP plus Mg to the plus 2 power was associated with a reciprocal increase in (32P) labeled HMG-CoA reductase with a cytosolic phosphatase resulted in a time dependent reciprocal release of (32P) protein-bound radioactivity and reactivation of enzyme activity. Phosphorylation of HMG-CoA reductase was confirmed by immunoprecipitation of (32P) HMG-CoA reductase with an anti-serum prepared against purified reductase. The existence of HMG-CoA reductase in an interconvertible active-inactive form of the enzyme provides a mechanism for the rapid short-term regulation of the pathway for cholesterol biosynthesis.